Cancer is characterized by abnormal regulation of cell growth, a process that ultimately depends on the correct expression and regulation of a large number of genes by transcription factors that can act as oncoproteins and tumor suppressors. We developed an inducible transformation model in which a transient inflammatory signal causes a stable non-transformed human breast epithelial cell line to undergo an epigenetic switch to a stable transformed state that includes a population of cancer stem cells (CSCs). The epigenetic switch is mediated by an inflammatory feedback loop consisting of transcription factors, miRNAs, and target genes that are oncogenes or tumor suppressors; this pathway is relevant for many forms of human cancer. We showed that CSCs and their non-stem cancer cell counterparts in the transformed population are not epigenetically distinct, but rather exist in a dynamic equilibrium involving interleukin 6 and an integrated transcriptional regulatory circuit that acts as a bistable switch. Lastly, we demonstrated that 3 transcriptional co-activator (-catenin, YAP/TAZ, S100A9/A8) that, respectively, are the ultimate targets of the Wnt, Hippo, and calcium signaling pathways, are critical for transformation. The central goal of this proposal is to elucidate, on a whole-genome scale, the transcriptional regulatory circuits involved in cellular transformation and CSC formation, neither of which have been investigated in this fashion. First, we will perform genetic (loss of function via siRNA or CRISPR followed by RNA-seq) and ChIP-seq experiments on candidate transcription factors we have already identified to determine where the factors bind in the genome and what genes they regulate. The results will be integrated into transcriptional regulatory circuits. Second, we will identify direct and indirec targets of co-activators (-catenin, YAP/TAZ, S100A9/A8) and integrate the results with those of the DNA-binding transcription factors. In addition, we identified WDR77 and the arginine methylase PRMT5 as interacting with -catenin as well as components of the polyadenylation machinery as interacting with YAP/TAZ; we will examine the molecular mechanisms by which these interacting proteins mediate their effects on gene expression and transformation. Third, using novel conceptual approaches based on mRNA profiles mediated by oncogenic and non-oncogenic protein derivatives or by different signaling molecules, we will identify oncogenically relevant targets. As a complement, we will use our high-throughput transformation assay to perform genome-scale genetic screens for genes important for transformation. The identified genes will be integrated into the transcriptional circuitry elucidated in aims 1 and 2. Fourth, the regulatory circuits derived from these results will be validated in other cancer cell types and by gene expression patterns in cancer patient samples. In summary, this tightly integrated set of genetic and functional genomic experiments on an inducible model of transformation and CSC formation will shed new light on fundamental issues in cancer progression at the molecular level, and new pathways and targets for therapy might be identified.